Arg660Ser mutation in Thermus acquaticus DNA polymerase I suppresses T→C transitions

Implication of wobble base pair formation at the nucleotide incorporation step

Katsushi Yoshida, Aki Tosaka, Hiroyuki Kamiya, Takashi Murate, Hiroshi Kasai, Yuji Nimura, Masanori Ogawa, Shonen Yoshida, Motoshi Suzuki

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

We examined the replication fidelity of an Arg660Ser (R660S) mutant of Thermus aquaticus DNA polymerase I (Taq pol I). In a forward mutation assay, R660S showed a marked reduction in T→C transitions, one of the most frequent errors made by the wild-type enzyme. Steady-state kinetics showed that R660S discriminates against dGTP incorporation at a template T 13-fold better than the wild-type. R660S was also 3.2-fold less efficient than the wild-type at extending a T:dG mismatch. These results indicate that R660S has enhanced fidelity during incorporation and extension, which reduces its T→C transition frequency. Interestingly, R660S also discriminated correct from incorrect nucleotides at the incorporation step of C:dATP, A:dATP, G:dATP and C:8-OH-dGTP mispairs 28-, 6.0-, 4.1- and 6.8-fold better, respectively, than the wild-type, although it may not always be as accurate as the wild-type at the extension step. A structural model suggests that Arg660 may participate in two interactions that influence fidelity; the guanidinium group of Arg660 might interact with the incoming guanine base at the major groove and it might compete for forming another interaction with the primer terminus. Substituting Arg with Ser may eliminate or alter these interactions and destabilize the closed complex with incorrect substrates. Our data also suggest that T:dGTP and C:dATP base pairs form 'wobble' structures at the incorporation step of Taq pol I.

Original languageEnglish
Pages (from-to)4206-4214
Number of pages9
JournalNucleic Acids Research
Volume29
Issue number20
Publication statusPublished - 15-10-2001
Externally publishedYes

Fingerprint

Thermus
DNA Polymerase I
Base Pairing
Nucleotides
Taq Polymerase
Mutation
Structural Models
Guanidine
Guanine
Enzymes
deoxyguanosine triphosphate
hydroxide ion
8-hydroxy-dGTP

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Yoshida, Katsushi ; Tosaka, Aki ; Kamiya, Hiroyuki ; Murate, Takashi ; Kasai, Hiroshi ; Nimura, Yuji ; Ogawa, Masanori ; Yoshida, Shonen ; Suzuki, Motoshi. / Arg660Ser mutation in Thermus acquaticus DNA polymerase I suppresses T→C transitions : Implication of wobble base pair formation at the nucleotide incorporation step. In: Nucleic Acids Research. 2001 ; Vol. 29, No. 20. pp. 4206-4214.
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abstract = "We examined the replication fidelity of an Arg660Ser (R660S) mutant of Thermus aquaticus DNA polymerase I (Taq pol I). In a forward mutation assay, R660S showed a marked reduction in T→C transitions, one of the most frequent errors made by the wild-type enzyme. Steady-state kinetics showed that R660S discriminates against dGTP incorporation at a template T 13-fold better than the wild-type. R660S was also 3.2-fold less efficient than the wild-type at extending a T:dG mismatch. These results indicate that R660S has enhanced fidelity during incorporation and extension, which reduces its T→C transition frequency. Interestingly, R660S also discriminated correct from incorrect nucleotides at the incorporation step of C:dATP, A:dATP, G:dATP and C:8-OH-dGTP mispairs 28-, 6.0-, 4.1- and 6.8-fold better, respectively, than the wild-type, although it may not always be as accurate as the wild-type at the extension step. A structural model suggests that Arg660 may participate in two interactions that influence fidelity; the guanidinium group of Arg660 might interact with the incoming guanine base at the major groove and it might compete for forming another interaction with the primer terminus. Substituting Arg with Ser may eliminate or alter these interactions and destabilize the closed complex with incorrect substrates. Our data also suggest that T:dGTP and C:dATP base pairs form 'wobble' structures at the incorporation step of Taq pol I.",
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Arg660Ser mutation in Thermus acquaticus DNA polymerase I suppresses T→C transitions : Implication of wobble base pair formation at the nucleotide incorporation step. / Yoshida, Katsushi; Tosaka, Aki; Kamiya, Hiroyuki; Murate, Takashi; Kasai, Hiroshi; Nimura, Yuji; Ogawa, Masanori; Yoshida, Shonen; Suzuki, Motoshi.

In: Nucleic Acids Research, Vol. 29, No. 20, 15.10.2001, p. 4206-4214.

Research output: Contribution to journalArticle

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T1 - Arg660Ser mutation in Thermus acquaticus DNA polymerase I suppresses T→C transitions

T2 - Implication of wobble base pair formation at the nucleotide incorporation step

AU - Yoshida, Katsushi

AU - Tosaka, Aki

AU - Kamiya, Hiroyuki

AU - Murate, Takashi

AU - Kasai, Hiroshi

AU - Nimura, Yuji

AU - Ogawa, Masanori

AU - Yoshida, Shonen

AU - Suzuki, Motoshi

PY - 2001/10/15

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N2 - We examined the replication fidelity of an Arg660Ser (R660S) mutant of Thermus aquaticus DNA polymerase I (Taq pol I). In a forward mutation assay, R660S showed a marked reduction in T→C transitions, one of the most frequent errors made by the wild-type enzyme. Steady-state kinetics showed that R660S discriminates against dGTP incorporation at a template T 13-fold better than the wild-type. R660S was also 3.2-fold less efficient than the wild-type at extending a T:dG mismatch. These results indicate that R660S has enhanced fidelity during incorporation and extension, which reduces its T→C transition frequency. Interestingly, R660S also discriminated correct from incorrect nucleotides at the incorporation step of C:dATP, A:dATP, G:dATP and C:8-OH-dGTP mispairs 28-, 6.0-, 4.1- and 6.8-fold better, respectively, than the wild-type, although it may not always be as accurate as the wild-type at the extension step. A structural model suggests that Arg660 may participate in two interactions that influence fidelity; the guanidinium group of Arg660 might interact with the incoming guanine base at the major groove and it might compete for forming another interaction with the primer terminus. Substituting Arg with Ser may eliminate or alter these interactions and destabilize the closed complex with incorrect substrates. Our data also suggest that T:dGTP and C:dATP base pairs form 'wobble' structures at the incorporation step of Taq pol I.

AB - We examined the replication fidelity of an Arg660Ser (R660S) mutant of Thermus aquaticus DNA polymerase I (Taq pol I). In a forward mutation assay, R660S showed a marked reduction in T→C transitions, one of the most frequent errors made by the wild-type enzyme. Steady-state kinetics showed that R660S discriminates against dGTP incorporation at a template T 13-fold better than the wild-type. R660S was also 3.2-fold less efficient than the wild-type at extending a T:dG mismatch. These results indicate that R660S has enhanced fidelity during incorporation and extension, which reduces its T→C transition frequency. Interestingly, R660S also discriminated correct from incorrect nucleotides at the incorporation step of C:dATP, A:dATP, G:dATP and C:8-OH-dGTP mispairs 28-, 6.0-, 4.1- and 6.8-fold better, respectively, than the wild-type, although it may not always be as accurate as the wild-type at the extension step. A structural model suggests that Arg660 may participate in two interactions that influence fidelity; the guanidinium group of Arg660 might interact with the incoming guanine base at the major groove and it might compete for forming another interaction with the primer terminus. Substituting Arg with Ser may eliminate or alter these interactions and destabilize the closed complex with incorrect substrates. Our data also suggest that T:dGTP and C:dATP base pairs form 'wobble' structures at the incorporation step of Taq pol I.

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